Interpretive Summary: Some wild birds that live in water habitats can be infected by avian influenza (AI) viruses in nature. Detecting these AI virus infections is difficult using traditional virus isolation or genetic detection technologies. We examined a new test, called blocking enzyme linked immunosorbent assay (bELISA), to detect antibodies in birds which indicates a past infection with AI virus. In our experimental samples, this bELISA was better than the older agar gel immunodiffusion test at determining which birds had been infected with either low or high pathogenicity AI viruses. This new test will assist bird ecologists in studying AI virus ecology in wild birds.

Technical Abstract:
Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoir for avian influenza (AI) viruses. Traditionally, AI surveillance in wild birds has relied on virus detection strategies including virus isolation and polymerase chain reaction. To evaluate the efficacy of a commercial blocking enzyme-linked immunosorbent assay (bELISA) and the agar gel immunodiffusion (AGID) assay for detection of antibodies to AI virus in wild birds, we tested 285 serum samples from various wild avian species that were experimentally infected with AI viruses. Included in these samples were 182 samples from birds with confirmed AI infections (125 infected with low pathogenic avian influenza (LPAI) viruses and 57 with highly pathogenic avian influenza (HPAI) viruses) and 103 samples from birds that were uninfected, negative controls. The sensitivities of the bELISA and AGID tests were 0.825 (95% CI: 0.701-0.913) and 0.681 (0.608-0.748), respectively. Both tests had an estimated specificity of 1.00 (95% CI: 0.965-1.00). The bELISA was significantly more sensitive than the AGID for both LPAI- and HPAI-infected birds. Both assays, however, had a higher sensitivity for HPAI-infected birds than LPAI infected. These results demonstrate the potential utility of the bELISA for detection of antibodies to both LPAI and HPAI viruses in multiple avian species representing 16 genera and five avian orders. Additional studies are warranted to further evaluate the utility of the bELISA assay in the field.